Abstract Multi-drug resistant TB (MDR-TB) is caused by Mycobacterium tuberculosis (Mtb) strains that are resistant to two front-line antibiotics, isoniazid (INH) and rifampin (RIF), in the recommended TB treatment regimen. The World Health Organization (WHO) estimates the prevalence of MDR-TB at approximately 50 million people worldwide, expanding by nearly 500,000 new cases each year. A more alarming development is the increase in and global distribution of extremely drug- resistant TB (XDR-TB), defined as Mtb resistant to INH, RIF and key second-line drugs. Early recognition of patients with M/XDR-TB and selection of appropriate antibiotics to which their isolates are susceptible would improve patient outcomes and assist in TB control efforts. While culturing of microorganisms to determine viability remains the gold standard for infectious disease diagnostics and phenotypic antibiotic susceptibility test (AST), the slow growth of Mtb delays AST results beyond practical utility for patient management or infection control. There are presently no satisfactory options for early, rapid (< 24 hr.), and sensitive detection of Mtb antibiotic resistance. Thus, there is a desperate need to identify a rapid diagnostic AST to prevent drug failure, relapse, and death from M/XDR-TB. Sequella developed a rapid (<1 day), relatively sensitive (?102 colony forming units), test to interrogate the metabolic potential of clinical Mtb isolates without culture when exposed to TB drugs. Recombinant phage engineered to contain the B-SMART? cassette take over the metabolism of Mtb and immediately direct the cell to synthesize multiple copies of a unique nucleic acid sequence not otherwise present in either the phage or Mtb. Antibiotics reduce B-SMARTTM signal because they interfere with cellular metabolism (transcription and translation), thus phage are not able to produce the signal and the readout is a phenotypic characterization of drug susceptibility. B-SMART? signal sequence is optimized for nucleic acid amplification (NAA) testing and can be detected by any NAA method. This R21 proposal will improve the sensitivity of the phage used in B-SMART? by using a cutting- edge synthetic genomics approach to improve its signal to noise ratio and test the optimized B- SMART? in Mtb clinical isolates. Once the phage is optimized and we test the sensitivity of the assay with the various TB drugs in a research laboratory setting, we will develop a clinical laboratory protocol in a subsequent application for 1) detection of live Mtb in patient sputum samples, 2) use in either centralized laboratories or a point-of-care setting, or both, 3) validation the B-SMART? AST using FDA guidelines, and 4) preparation for commercial launch.